The purpose of this BRP is to develop and evaluate technology for three-dimensional imaging of cells in the living eye, and to use this novel technology to understand changes in cell layers associated with the most common diseases leading to world-wide blindness, including age-related macular degeneration and glaucoma. Partners at four institutions will contribute to instrumentation that combines adaptive optics (AO), providing high lateral resolution, with optical coherence tomography (OCT), providing high axial resolution. The lead institution is the University of California, Davis (John S. Werner, PI). Both at UC Davis and at Indiana University (Donald T. Miller, site PI), AO-OCT instrumentation will be developed and tested. Duke University (Joseph Izatt, site PI) will develop novel OCT approaches that will be incorporated, while Lawrence Livermore National Laboratory (Scot Olivier, site PI) will provide cutting-edge advances in AO. These AO-OCT instruments will permit human in vivo imaging with sufficient resolution and contrast to visualize the smallest of cells in the human retina. In the previous project period we have used AO-OCT to create volume images of structures previously only visible with histology, including the photoreceptor outer segments, Fibers of Henle, individual optic nerve fiber bundles, detailed structures within drusen of macular degeneration patients, and fine structure of the lamina cribosa of the optic nerve. In this renewal, we propose to solve new technical issues to more fully tap the potential and functionality of AO-OCT. The engineering goals are directed toward increasing contrast of cellular structures by use of autofluoresence, birefringence, the incorporation of extreme AO techniques, and functional changes associated with blood perfusion, hemoglobin oxygen saturation in the retinal vasculature as well as electrical changes in neuronal cell volumes. Considerable effort will be devoted to three-dimensional visualization and segmentation over retinal volumes of larger lateral and axial extent than previously possible. The engineering goals have parallel clinical aims for improving our understanding of the cellular changes associated with markers for age-related macular degeneration and glaucoma. These advanced imaging techniques will be deployed for evaluating the changes in the retina and optic nerve resulting from novel therapies for rescuing retinal layers from the leading causes of blindness.